Toxin-Eating Bacteria and Bioremediation

ABSTRACT

Bacteria that can use antibiotics as a carbon source are provided. Methods and bacteria useful for bioremediation are also provided.

PRIORITY INFORMATION

This application is a divisional of U.S. application Ser. No. 12/579,696filed Oct. 15, 2009, which claims priority to U.S. Provisional PatentApplication No. 61/105,614, filed on Oct. 15, 2008, all of which arehereby incorporated herein by reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with government support under DE-FG02-03ER63445(T-103693) awarded by the U.S. Department of Energy. The government hascertain rights in the invention.

BACKGROUND

The seemingly unchecked spread of multiple antibiotic resistance inclinically relevant pathogenic microbes is alarming. Furthermore, asignificant environmental reservoir of antibiotic resistancedeterminants, termed the antibiotic resistome, has been discovered(Riesenfeld et al. (2004) Environmental Microbiology 6:981; D'Costa etal. (2006) Science 311:374). The primary microbial antibiotic resistancemechanisms include efflux pumps, target gene-product modifications, andenzymatic inactivation of the antibiotic compound (Walsh (2000) Nature406:775; Alekshun and Levy (2007) Cell 128:1037). Many of the mechanismsare common to several species of pathogens and spread by lateral genetransfer (Davies (1994) Science 264:375). While many bacteria growing inextreme environments (Davies (1994) Science 264:375) and capable ofdegrading toxic substrates (McAllister et al. (1996) Biodegradation 7:1)have been previously reported, only a few organisms have been shown tosubsist on a limited number of antibiotic substrates (Kameda et al.(1961) Nature 191:1122; Johnsen (1977) Archives of Microbiology 115:271;Abdelm et al. (1961) Nature 189:775).

SUMMARY

The present invention is based in part on the surprising discovery thatthe microbiome (e.g., of soil and/or water) includes a significantreservoir of bacteria capable of subsisting on antibiotics. Clonalbacterial isolates were obtained from eleven diverse soils which werecapable of utilizing one of 18 different antibiotics as the sole carbonsource. The 18 antibiotics comprised of natural, semi-synthetic andsynthetic compounds and included all major bacterial target classes.

Accordingly, in certain exemplary embodiments, a method of reducing alevel of one or more antibiotics from an antibiotic-contaminatedsubstance comprising culturing an organism that can utilize the one ormore antibiotics as a carbon source (e.g., a sole carbon source) in thepresence of the antibiotic-contaminated substance for a sufficientamount of time to reduce the level of one or more antibiotics from theantibiotic-contaminated substance is provided. In certain aspects, theorganism is a bacterium. In other aspects, one or more antibiotics arefrom the antibiotic class including one or more of pyrimidinederivative, sulfonamide, quinolone, glycopeptides, beta-lactam,amphenicols, aminoglycoside and amino acid derivative. In yet otheraspects, one or more antibiotics are selected from the group includingone or more of chloramphenicol, penicillin G, vancomycin, carbenicillin,ciprofloxacin, mafenide, kanamycin, sisomicin, amikacin, trimethropin,D-cycloserine, gentamicin, dicloxacillin, nalidixic acid, thiamphenicol,levofloxacin, sulfamethizole and sulfisoxazole. In still other aspects,the antibiotic-contaminated substance is one or more of contaminatedsoil, contaminated water and a contaminated work surface (e.g., in ahospital, a clinic, a laboratory or the like).

In certain exemplary embodiments, a bacterium that can use one or moreantibiotics as a carbon source (e.g., a sole carbon source) is provided.The bacterium has a 16S nucleic acid sequence comprising a GenBankAccession Number selected from one or more of EU515334, EU515335,EU515336, EU515337, EU515338, EU515339, EU515400, EU515401, EU515402,EU515403, EU515404, EU515405, EU515406, EU515407, EU515408, EU515409,EU515410, EU515411, EU515412, EU515413, EU515414, EU515415, EU515416,EU515417, EU515418, EU515419, EU515420, EU515421, EU515422, EU515423,EU515424, EU515425, EU515426, EU515427, EU515428, EU515429, EU515430,EU515431, EU515432, EU515433, EU515434, EU515435, EU515436, EU515437,EU515438, EU515439, EU515440, EU515441, EU515442, EU515443, EU515444,EU515445, EU515446, EU515447, EU515448, EU515449, EU515450, EU515451,EU515452, EU515453, EU515454, EU515455, EU515456, EU515457, EU515458,EU515459, EU515460, EU515461, EU515462, EU515463, EU515464, EU515465,EU515466, EU515467, EU515468, EU515469 EU515470, EU515471, EU515472,EU515473, EU515474, EU515475, EU515476, EU515477, EU515478, EU515479,EU515480, EU515481, EU515482, EU515483, EU515484, EU515485, EU515486,EU515487, EU515488, EU515489, EU515490, EU515491, EU515492, EU515493,EU515494, EU515495, EU515496, EU515497, EU515498, EU515499, EU515500,EU515501, EU515502, EU515503, EU515504, EU515505, EU515506, EU515507,EU515508, EU515509, EU515510, EU515511, EU515512, EU515513, EU515514,EU515515, EU515516, EU515517, EU515518, EU515519, EU515520, EU515521,EU515522, EU515523, EU515524, EU515525, EU515526, EU515527, EU515528,EU515529, EU515530, EU515531, EU515532, EU515533, EU515534, EU515535,EU515536, EU515537, EU515538, EU515539, EU515540, EU515541, EU515542,EU515543, EU515544, EU515545, EU515546, EU515547, EU515548, EU515549,EU515550, EU515551, EU515552, EU515553, EU515554, EU515555, EU515556,EU515557, EU515558, EU515559, EU515560, EU515561, EU515562, EU515563,EU515564, EU515565, EU515566, EU515567, EU515568, EU515569, EU515570,EU515571, EU515572, EU515573, EU515574, EU515575, EU515576, EU515577,EU515578, EU515579, EU515580, EU515581, EU515582, EU515583, EU515584,EU515585, EU515586, EU515587, EU515588, EU515589, EU515590, EU515591,EU515592, EU515593, EU515594, EU515595, EU515596, EU515597, EU515598,EU515599, EU515600, EU515601, EU515602, EU515603, EU515604, EU515605,EU515606, EU515607, EU515608, EU515609, EU515610, EU515611, EU515612,EU515613, EU515614, EU515615, EU515616, EU515617, EU515618, EU515619,EU515620, EU515621, EU515622 and EU515623. In certain aspects, one ormore antibiotics are from the antibiotic class including one or more ofpyrimidine derivative, sulfonamide, quinolone, glycopeptides,beta-lactam, amphenicols, aminoglycoside and amino acid derivative. Inother aspects, one or more antibiotics include one or more ofchloramphenicol, penicillin G, vancomycin, carbenicillin, ciprofloxacin,mafenide, kanamycin, sisomicin, amikacin, trimethropin, D-cycloserine,gentamicin, dicloxacillin, nalidixic acid, thiamphenicol, levofloxacin,sulfamethizole and sulfisoxazole.

In certain exemplary embodiments, a method of removing one or moreantibiotics from an antibiotic-contaminated substance, comprisingculturing a bacterium described above in the presence of theantibiotic-contaminated substance for a sufficient amount of time toreduce the level of one or more antibiotics from theantibiotic-contaminated substance is provided. In certain aspects, oneor more antibiotics are from the antibiotic class including one or moreof pyrimidine derivative, sulfonamide, quinolone, glycopeptides,beta-lactam, amphenicols, aminoglycoside and amino acid derivative. Inother aspects, one or more antibiotics are selected from the groupincluding one or more of chloramphenicol, penicillin G, vancomycin,carbenicillin, ciprofloxacin, mafenide, kanamycin, sisomicin, amikacin,trimethropin, D-cycloserine, gentamicin, dicloxacillin, nalidixic acid,thiamphenicol, levofloxacin, sulfamethizole and sulfisoxazole. In yetother aspects, the antibiotic-contaminated substance is one or more ofcontaminated soil, contaminated water and a contaminated work surface(e.g., in a hospital, a clinic, a laboratory or the like.

Further features and advantages of certain embodiments of the presentinvention will become more fully apparent in the following descriptionof the embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIGS. 1A-1B graphically depict clonal bacterial isolates subsisting onantibiotics. (A) Heat-map illustrating growth results from allcombinations of 11 soils by 18 antibiotics, where blue squares representsuccessful isolation of bacteria from a given soil that are able toutilize that antibiotic as sole carbon source at 1 g/L. Soil sampleslabeled F1-3 were from farm soils and U1-3 were from urban soils. Soilsamples P1-5 were from pristine soils, collected from non-urban areaswith minimal human exposure over the last 100 years (Table 2). (B) Highperformance liquid chromatography (HPLC) traces at 214 nm ofrepresentative penicillin and carbenicillin catabolizing clonal isolatesand corresponding un-inoculated media controls for different time pointsover 20 or 28 days of growth, respectively.

FIG. 2 graphically depicts the phylogenetic distribution of bacterialisolates subsisting on antibiotics. 16S ribosomal DNA (rDNA) wassequenced from antibiotic catabolizing clonal isolates using universalbacterial rDNA primers. High-quality, non-chimeric sequences wereclassified using Greengenes (DeSantis et al. (2006) Applied andEnvironmental Microbiology 72:5069), with consensus annotations from RDP(Cole et al. (2007) Nucl. Acids Res. 35:D169) and NCBI taxonomies (D. L.Wheeler et al. (2000) Nucl. Acids Res. 28:10). Phylogenetic trees wereconstructed using the neighbor joining algorithm in ARB (W. Ludwig etal. (2004) Nucl. Acids Res. 32:1363) using the Greengenes aligned 16SrDNA database. Placement in the tree was confirmed by comparingautomated Greengenes taxonomy to the annotated taxonomies of nearestneighbors of each sequence in the aligned database. Branches of the treeare color-coded by bacterial orders, and clonal isolates represented assquares. Accession numbers of certain of these bacterial isolates thathave been deposited are from EU515334 to EU515623 (GenBank), and arehereby incorporated by reference in their entirety.

FIGS. 3A-3C graphically depict antibiotic resistance profiling of 75clonal isolates capable of subsisting on antibiotics. (A) Heat mapillustrating the resistance profiles of a representative subset of 75clonal isolates capable of utilizing antibiotics as sole carbon source(Table 3). Resistance was determined as growth after 4 days at 22° C. inLuria Broth media containing 20 mg/liter antibiotic (top panel) and 1g/liter antibiotic (bottom panel). (B) Percentage of clonal isolatesresistant to each of the 18 antibiotics. Antibiotics are color coded byclass, the full height of each bar corresponds to the percentage ofclonal isolates resistant at 20 mg/liter and the solid colored sectionof each bar corresponds to the percentage of clonal isolates resistantat 1 g/liter. (C) Histogram depicting the distribution of the number ofantibiotics that the clonal isolates were resistant to at 20 mg/liter(top panel) and 1 g/liter (bottom panel).

FIGS. 4A-4B graphically depict the distribution of antibioticcatabolizing bacterial isolates with respect to antibiotics and soil.(A) Number of antibiotic catabolizing bacteria isolated from 11 soilscolor-coded by antibiotic class catabolized. (B) Percentage of soilscontaining antibiotic catabolizing bacteria, color-coded by chemicalorigin of antibiotic.

FIG. 5 schematically depicts the phylogenetic distribution of bacterialisolates subsisting on antibiotics. Full set of bacteria subsisting onantibiotics is displayed in the centre, with branches color-coded bybacterial orders, and clonal isolates represented as squares. Subsetscomprising clonal isolates catabolizing each antibiotic are representedas trees around the periphery, grouped by antibiotic class. 16Sribosomal DNA (rDNA) was sequenced from antibiotic catabolizing clonalisolates using universal bacterial rDNA primers. High-quality,non-chimeric sequences were classified using Greengenes (DeSantis et al.(2006) Applied and Environmental Microbiology 72:5069), with consensusannotations from RDP (Cole et al. (2007) Nucleic Acids Res 35:D169) andNCBI taxonomies (Wheeler et al. (2000) Nucleic Acids Res 28:10).Phylogenetic trees were constructed using the neighbor-joining algorithmin ARB (Ludwig et al. (2004) Nucleic Acids Res 32:1363) using theGreengenes aligned 16S rDNA database. Placement in the tree wasconfirmed by comparing automated Greengenes taxonomy to the annotatedtaxonomies of nearest neighbors of each sequence in the aligneddatabase. The phylogenetic distributions of species isolated fromdifferent antibiotics as sole carbon source exhibit some interestingtrends. For instance, the fluoroquinolone antibiotics, ciprofloxacin andlevofloxacin, have similar phylogenetic distributions, as do theaminoglycoside antibiotics, gentamycin and amikacin, but the two setsare notably different from each other. Interestingly, the orders ofbacteria subsisting on amikacin appear more similar to gentamycin thankanamycin despite amikacin being a semi synthetic kanamycin derivative.

FIGS. 6A-6C depict mass spectrometry analysis of growth media frompenicillin subsisting bacterial culture. (A) Mass spectra of day 0growth media from penicillin culture with a major peak at m/z of 335.10corresponding exactly to the protonated penicillin G molecule. (B) Massspectra of day 4 growth media from penicillin culture with two majorpeaks at m/z values 353.11 and 309.12 corresponding to protonatedbenzylpenicilloic acid and benzylpenilloic acid, respectively. (C) Firststeps of a proposed penicillin G degradation pathway.

FIG. 7 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB2: Antibiotic Box 2; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 8 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB3: Antibiotic Box 3; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 9 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB4: Antibiotic Box 4; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 10 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB5: Antibiotic Box 5; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 11 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB6: Antibiotic Box 6; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 12 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB7: Antibiotic Box 7; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

FIG. 13 depicts a list of antibiotic catabolizing isolates described inFIG. 2. AIB8: Antibiotic Box 8; S*: section; YDM-TM: 1X YDM, tracemetals, pH 5.5; Extr: extraction; RT: room temperature.

DETAILED DESCRIPTION

Man-made chemicals are often used to clean up contaminated and/or toxicmaterials, which can be both costly and time-consuming. Microorganisms(e.g., bacteria) are a natural, inexpensive means for reducing and/oreliminating contamination and/or toxicity of a substance. Accordingly,in certain exemplary embodiments, antibiotic and/or toxin eatingmicroorganisms (e.g., bacteria) that can be produced using the methodsdescribed herein are provided. In certain aspects, a cell, cell lysate,cell extract, cell fraction, protein(s), polypeptide(s), isolatedantibiotic(s) or any combinations thereof from one or more antibioticand/or toxin eating microorganisms (e.g., bacteria) are incubated in thepresence of a contaminated substance to reduce or eliminatecontamination. In another aspect, antibiotic and/or toxin eatingbacteria are used in hybrid biological/chemical manufacturing ordecontamination systems where resistance to high levels of variouschemicals is helpful in the process engineering. A cell, cell lysate,cell extract, cell fraction, protein(s), polypeptide(s), isolatedantibiotic(s) or any combinations thereof from one or more antibioticand/or toxin eating microorganisms (e.g., bacteria) can be applied to acontaminated substance or a manufacturing system via aerosols, slurries,cleaning solutions, animal feeds, seeds, fertilizer and the like topartially or completely decontaminate the substance or manufacturingsystem.

As used herein, the terms “toxin-eating bacterium” and “toxin-eatingbacteria” refer to bacteria that can use one or more toxins and/orcontaminants as a carbon source(s) or as the sole carbon source tosupport growth. As used herein, the terms “antibiotic-eating bacterium”and “antibiotic-eating bacteria” refer to bacteria that can use one ormore antibiotics as a carbon source(s) or as the sole carbon source tosupport growth.

In certain exemplary embodiments, one or more toxin-eating bacteriadescribed herein are used for bioremediation of one or more contaminantsfrom a variety of environments such as, e.g., earth (e.g., sand, soil,rocks, any combination thereof and the like), water (e.g., springs,lakes, brooks, streams, rivers, bays, estuaries, seas, oceans and thelike), air, manmade surfaces (e.g., medical facilities, instruments,service salons, makeup counters etc.) and the like. As used herein, theterm “bioremediation” refers to the ability of one or more bacteriadescribed herein to remove or reduce the levels of one or morecontaminants from an environment.

As used herein, the terms “toxic environment” and “contaminatedsubstance” refer to an environment or substance, respectively, thatcontains one or more adverse compound(s) and/or physical condition(s)that can inhibit growth, inhibit productivity and/or lead to the deathof one or more microorganisms exposed to the compound(s) and/or physicalcondition(s). A toxic environment includes, but is not limited to, thefollowing: the presence of inhibitory compounds (e.g., antibiotics,radioactive compounds, heavy metals and the like) high or low salinity,extreme temperatures (e.g., high temperature (e.g., in thermal vents)and/or cold temperature (e.g., in icy conditions), water scarcity,darkness, light, catalytic products (e.g., cell waste, alcohol and thelike) and the like. For example, a toxic environment can include thepresence of a concentration (e.g., high or low concentrations) of acompound and/or a condition that is considered non-toxic to themicroorganism in typical concentrations and/or in typical conditions, aswell as the presence of a compound or a physical condition that would betypically considered to be detrimental to the organism.

In certain embodiments, the toxicity (of a toxic environment) orcontamination (of a contaminated substance) is eliminated or reduced tonon-toxic or non-contaminated levels. In certain aspects, the toxicityand/or contamination is reduced by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more.

In certain exemplary embodiments, DNA fragments that can be used in amicroorganism to decrease toxicity and/or contamination of a substanceare provided. In certain aspects, the identification of useful DNAfragments occurs by introducing a diverse library of DNA fragments intoa clonal population of the production microorganism creating apopulation of cells harboring different DNA fragments. The population ofmicroorganisms harboring the large DNA fragment library is subjected togrowth in the presence of high concentration of the inhibitor(s) whichwould normally suppress growth of the host organism. If a host cell inthe population contains a DNA fragment which encodes for resistance to,e.g., high concentrations of inhibitor(s) (e.g., one or moreantibiotics), the cell will selectively grow and can be identified. TheDNA fragment that enabled the cell to tolerate the inhibitor can then beisolated, characterized and subsequently introduced into the productionmicroorganism improving its catalytic productivity in the presence ofthe inhibitor.

As used herein, the term “organism” includes, but is not limited to, ahuman, a non-human primate, a cow, a horse, a sheep, a goat, a pig, adog, a cat, a rabbit, a mouse, a rat, a gerbil, a frog, a toad, a fish(e.g., D. rerio) a roundworm (e.g., C. elegans) and any transgenicspecies thereof. The term “organism” further includes, but is notlimited to, a yeast (e.g., S. cerevisiae) cell, a yeast tetrad, a yeastcolony, a bacterium, a bacterial colony, a virion, virosome, virus-likeparticle and/or cultures thereof, and the like.

As used herein, the terms “microorganism” and “microbe” refer to tinyorganisms. Most microorganisms and microbes are unicellular, althoughsome multicellular organisms are microscopic, while some unicellularprotists and bacteria (e.g., T. namibiensis) called are visible to thenaked eye. Microorganisms and microbes include, but are not limited to,bacteria, fungi, archaea and protists, microscopic plants, and animals(e.g., plankton, the planarian, the amoeba) and the like.

Certain aspects of the invention pertain to vectors, such as, forexample, expression vectors, containing a nucleic acid encoding one ormore bipolar cell-specific regulatory sequences. As used herein, theterm “vector” refers to a nucleic acid sequence capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, wherein additional DNA segments can be ligated into theviral genome. By way of example, but not of limitation, a vector of theinvention can be a single-copy or multi-copy vector, including, but notlimited to, a BAC (bacterial artificial chromosome), a fosmid, a cosmid,a plasmid, a suicide plasmid, a shuttle vector, a P1 vector, an episome,YAC (yeast artificial chromosome), a bacteriophage or viral genome, orany other suitable vector. The host cells can be any cells, includingprokaryotic or eukaryotic cells, in which the vector is able toreplicate.

Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors.” In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of interest (e.g., a nucleic acid sequence from a microorganism) ina form suitable for expression of the nucleic acid in a host cell, whichmeans that the recombinant expression vectors include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operatively linked to the nucleic acid sequenceto be expressed. Within a recombinant expression vector, “operablylinked” is intended to mean that the nucleotide sequence of interest ispresent in the vector in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences).

It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or portions thereof,including fusion proteins or portions thereof, encoded by nucleic acidsas described herein.

In certain exemplary embodiments, a nucleic acid described herein isexpressed in bacterial cells using a bacterial expression vector suchas, e.g., a fosmid. A fosmid is a cloning vector that is based on thebacterial F-plasmid. The host bacteria will typically only contain onefosmid molecule, although an inducible high-copy on can be included suchthat a higher copy number can be obtained (e.g., pCC1FOS™, pCC2FOS™).Fosmid libraries are particularly useful for constructing stablelibraries from complex genomes. Fosmids and fosmid library productionkits are commercially available (EPICENTRE® Biotechnologies, Madison,Wis.). For other suitable expression systems for both prokaryotic andeukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, oneor more bipolar cell-specific regulatory elements and/or portion(s)thereof can be reproduced in bacterial cells such as E. coli, virusessuch as retroviruses, insect cells, yeast or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells). Other suitable hostcells are known to those skilled in the art.

Delivery of nucleic acid sequences described herein (e.g., vector DNA)can be by any suitable method in the art. For example, delivery may beby injection, gene gun, by application of the nucleic acid in a gel,oil, or cream, by electroporation, using lipid-based transfectionreagents, or by any other suitable transfection method.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection (e.g., usingcommercially available reagents such as, for example, LIPOFECTIN®(Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE® (Invitrogen),FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™(Polyplus-transfection Inc., New York, N.Y.), EFFECTENE® (Qiagen,Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), orelectroporation (e.g., in vivo electroporation). Suitable methods fortransforming or transfecting host cells can be found in Sambrook, et al.(Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring harborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

In certain exemplary embodiments, one or more host microorganismsdescribed herein are engineered with various isolation and/or safetyfeatures such as, e.g., novel genetic codes, broad restriction systems,extreme sensitivity to substances common in nature (e.g., UV light),dependency on lab metabolites uncommon in nature (e.g., diaminopimelicacid) and the like in order to decrease the spread of antibiotic and/ortoxin resistance gene(s) from one or more host cells. A non-limitingexample of a broad restriction system would be expression in the samecell endonucleases aimed at both the methylated and unmethylated formsof a DNA sequence (e.g., DpnI and DpnII aimed at G-mA-T-C and GATC).This would require the removal of all sites (GATC in the above example)throughout the host genome.

In certain exemplary embodiments, antibiotic and/or toxin eatingmicroorganisms (e.g., bacteria) are used to develop novel antibiotics.Novel antibiotics are useful for overcoming the multi-drug resistance(MDR) that is increasingly observed among pathogenic bacteria. Incertain exemplary aspects, antibiotic and/or toxin eating bacteria areused to manufacture novel antibiotics either harvested metagenomicallyfrom diverse natural microbial cells or engineered from combinatoriallibraries. Even the trace amounts need to detect biosynthesis of novelcompounds could be enough to kill the host (or put undesired pressure tobe unproductive).

Novel antibiotics can be manufactured, for example, by metagenomicharvesting from natural microbial cells or by engineering fromcombinatorial libraries. In certain exemplary embodiments, one or moremicroorganisms that are resistant to one or more compounds thattypically kill and/or inhibit the growth of the microorganism (e.g.,antibiotics, toxins and the like) are used in screening assays foridentifying modulators, i.e., candidate or test compounds or agents(e.g., antibodies, peptides, cyclic peptides, peptidomimetics, smallmolecules, small organic molecules, antibiotics or drugs) which kill orhave an inhibitory effect on the growth of one or more microorganismsare provided. In certain aspects, such screening assays can identifynovel antibiotics as well as antibiotics that are effective in killingor reducing the growth of one or more multiple antibiotic resistantmicroorganisms.

As used herein, the term “antibiotic” refers to a chemotherapeutic agent(e.g., an agent produced by microorganisms and/or synthetically) thathas the capacity to inhibit the growth of and/or to kill, one or moremicroorganisms (e.g., bacteria, fungi, parasites and the like) oraberrantly growing cells (e.g., tumor cells). As used herein,antibiotics are well-known to those of skill in the art. Classes ofantibiotics include, but are not limited to, aminoglycosides (e.g.,amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin,tobramycin, paromomycin and the like), ansamycins (e.g., geldanamycin,herbimycin and the like), carbacephem (e.g., loracarbef), carbapenems(e.g., ertapenem, doripenem, imipenem/cilastatin, meropenem and thelike) cephalosporins (e.g., first generation (e.g., cefadroxil,cefazolin, cefalotin, cefalexin and the like), second generation (e.g.,cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime and the like),third generation (e.g., cefixime, cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone and the like), fourth generation (e.g., cefepime and thelike) and fifth generation (e.g., ceftobiprole and the like)),glycopeptides (e.g., teicoplanin, vancomycin and the like), macrolides(e.g., azithromycin, clarithromycin, dirithromycin, erythromycin,roxithromycin, troleandomycin, telithromycin, spectinomycin and thelike), monobatams (e.g., aztreonam and the like), penicillins (e.g.,amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin,oxacillin, penicillin, piperacillin, ticacillin and the like),polypeptides (e.g., bacitracin, colistin, polymyxin B and the like)quinolones (e.g., ciprofloxacin, enoxacin, gatifloxacin, levofloxacin,lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin andthe like), sulfonamides (e.g., mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole,trimethoprim, trimethoprim-sulfamethoxazole and the like), tetracyclines(e.g., demeclocycline, doxycycline, minocycline, oxytetracycline,tetracycline and the like) and others (e.g., arsphenamine,chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin,fusidic acid, furazolidone, isoniazid, linezolid, metronidazole,mupirocin, nitrofurantoin, platensimycin, pyrazinamide,quinupristin/dalfopristin, rifampin, tinidazol and the like) (See, e.g.,Robert Berkow (ed.) The Merck Manual of Medical Information—HomeEdition. Pocket (September 1999), ISBN 0-671-02727-1).

In certain exemplary embodiments, assays for screening candidate or testcompounds (e.g., antibiotics) which bind to or modulate (e.g., kill orhave an inhibitory effect on the growth of) a microorganism areprovided. The test compounds of the present invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the “one-bead one-compound” librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

The candidate or test compound(s) described herein can be incorporatedinto pharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule or protein anda pharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

In certain exemplary embodiments, a pharmaceutical composition isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CREMOPHOREL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thecandidate or test compound(s) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: A binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic, acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant: such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the candidate or test compound(s) are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Nasal compositions generally include nasal sprays and inhalants. Nasalsprays and inhalants can contain one or more active components andexcipients such as preservatives, viscosity modifiers, emulsifiers,buffering agents and the like. Nasal sprays may be applied to the nasalcavity for local and/or systemic use. Nasal sprays may be dispensed by anon-pressurized dispenser suitable for delivery of a metered dose of theactive component. Nasal inhalants are intended for delivery to the lungsby oral inhalation for local and/or systemic use. Nasal inhalants may bedispensed by a closed container system for delivery of a metered dose ofone or more active components.

In one embodiment, nasal inhalants are used with an aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A non-aqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers maybe used to minimize exposing the agent to shear, which can result indegradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The candidate or test compound(s) can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, candidate or test compound(s) are prepared withcarriers that will protect them against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of candidate or test compound(s) canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% of the population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

Data obtained from cell culture assays and/or animal studies can be usedin formulating a range of dosage for use in humans. The dosage typicallywill lie within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In certain exemplary embodiments, a method for treatment of infection bya microorganism includes the step of administering a therapeuticallyeffective amount of an agent (e.g., one or more candidate or testcompounds) which modulates (e.g., kills and/or inhibits the growth of),one or more microorganisms to a subject. As defined herein, atherapeutically effective amount of agent (i.e., an effective dosage)ranges from about 0.001 to 30 mg/kg body weight, from about 0.01 to 25mg/kg body weight, from about 0.1 to 20 mg/kg body weight, or from about1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kgbody weight. The skilled artisan will appreciate that certain factorsmay influence the dosage required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of an inhibitor can include a singletreatment or, in certain exemplary embodiments, can include a series oftreatments. It will also be appreciated that the effective dosage ofinhibitor used for treatment may increase or decrease over the course ofa particular treatment. Changes in dosage may result from the results ofdiagnostic assays as described herein. The pharmaceutical compositionscan be included in a container, pack, or dispenser together withinstructions for administration.

In certain embodiments, monitoring the influence of agents (e.g., drugs,compounds) on the killing and/or inhibiting cell growth of one or moremicroorganisms can be applied not only in basic drug screening, but alsoin clinical trials. In certain exemplary embodiments, a method isprovided for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, antibody, peptidomimetic,protein, peptide, nucleic acid, small molecule, antibiotic or other drugcandidate identified by the screening assays described herein)comprising the steps of (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the levelof a microorganism in the preadministration sample; (iii) obtaining oneor more post-administration samples from the subject; (iv) detecting thelevel the microorganism in the post-administration samples; (v)comparing the level of microorganism in the pre-administration samplewith the level of microorganism in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease the effectiveness of the agent.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, tables, figures, andaccompanying claims.

Example I Bacteria Subsisting on Antibiotics

Antibiotics are a crucial line of defense against bacterial infections.Nevertheless, several antibiotics are natural products of microorganismsthat have as yet poorly appreciated ecological roles in the widerenvironment. Hundreds of soil bacteria with the capacity to grow onantibiotics as a sole carbon source were isolated. Of 18 antibioticstested, representing eight major classes of natural and syntheticorigin, 13-17 antibiotics supported growth of clonal bacteria from eachof 11 diverse soils. Bacteria subsisting on antibiotics are surprisinglyphylogenetically diverse and many are closely related to humanpathogens. Furthermore, each antibiotic consuming isolate is resistantto multiple antibiotics at clinically relevant concentrations. Thisphenomenon suggests this unappreciated reservoir of antibioticresistance determinants can contribute to the increasing levels ofmultiple antibiotic resistance in pathogenic bacteria.

Clonal bacterial isolates from 11 diverse soils (Table 2) which werecapable of utilizing one of 18 different antibiotics as the sole carbonsource were cultured. The 18 antibiotics comprised of natural,semi-synthetic and synthetic compounds of different ages and includedall major bacterial target classes. Every antibiotic tested was able tosupport bacterial growth (FIG. 1A and FIGS. 4A-4B). Notably, 6 out of 18antibiotics supported growth in all 11 soils, covering 5 of the 8classes of antibiotics tested. Appropriate controls were performed toensure that carbon source contamination of the source media or carbonfixation from the air were insignificant to this experiment (See ExampleII).

Clonal isolates capable of subsisting on penicillin and carbenicillinwere obtained from all the soils tested, and isolates from 9 out of 11soils that could subsist on dicloxacillin. Representative isolatescapable of growth on penicillin and carbenicillin were selected forsubsequent analysis by high performance liquid chromatography (HPLC)(See Example II). Removal of the antibiotics from the media was observedwithin 4 and 6 days, respectively (FIG. 1B). Mass spectrometry analysisof penicillin cultures is consistent with a penicillin catabolic pathway(Johnsen (1977) Archives of Microbiology 115:271) initiated byhydrolytic cleavage of the beta lactam ring, which is the dominant modeof clinical resistance to penicillin and related beta lactamantibiotics, followed by a decarboxylation step (FIGS. 6A-6C) (SeeExample II).

Bacteria were isolated from all the soils tested that grew onciprofloxacin (FIG. 1A), a synthetic fluoroquinolone and one of the mostwidely prescribed antibiotics. Clonal isolates capable of catabolizingthe other two synthetic quinolones tested, levofloxacin and nalidixicacid, were also isolated from a majority of the soils (FIG. 1A).Previous studies have highlighted the strong parallels betweenantibiotic resistance determinants harbored by soil dwelling microbesand human pathogens (Davies (1994) Science 264:375; Marshall (1998)Antimicrobial Agents and Chemotherapy 42:2215; D'Costa et al. (2007)Curr. Opin. Microbiol. 10:481). The lateral transfer of genes encodingthe enzymatic machinery responsible for subsistence on quinoloneantibiotics to human pathogens could introduce a novel resistancemechanism so far not observed in the clinic.

Phylogenetic profiling of the clonal isolates (See Example II) revealeda diverse set of species in the Proteobacteria (87%), Actinobacteria(7%) and Bacteroidetes (6%) (FIG. 2 and FIG S2). These phyla all includemany clinically relevant pathogens. Of the eleven orders represented,Burkholderiales constitute 41% of the species isolated. The other majororders (>5%) are: Pseudomonadales (24%), Enterobacteriales (13%),Actinomycetales (7%), Rhizobiales (7%), and Sphingobacteriales (6%).

Without intending to be bound by scientific theory, one explanation forthe widespread catabolism of both natural and synthetic antibiotics mayrelate to their organic sub-structures which are found in nature.Metabolic mechanisms exist for processing those sub-structures and mayallow for the utilization of the parent synthetic antibiotic molecule.It is interesting that more than half of the bacterial isolatesidentified in this study belong to the orders Burkholderiales andPseudomonadales. Organisms in these orders typically have large genomesof approximately 6-10 megabases, which has been suggested to bepositively correlated to their metabolic diversity and multipleantibiotic resistance (Projan (2007) Antimicrobial Agents andChemotherapy 51:1133). These organisms can be thought of as scavengers,capable of utilizing a large variety of single carbon sources as food(Parke and Gurian-Sherman (2001) Annual Review of Phytopathology39:225).

The magnitude of antibiotic resistance for a representative subset of 75clonal isolates was determined (Table 3). Each clonal isolate was testedfor resistance towards all 18 antibiotics used in the subsistenceexperiments at 20 mg/L and 1 g/L in rich media (See Example II). Theclonal isolates tested on average were resistant to 17 out of 18antibiotics at 20 mg/L, and 14 out of 18 antibiotics at 1 g/L (FIG. 3).Furthermore, for 74 of the 75 isolates, it was determined that if abacterial isolate was able to subsist on an antibiotic, it was alsoresistant to all antibiotics in that class at 20 mg/L.

Previous work showing that strains from the genus Streptomyces are onaverage resistant to 7-8 antibiotics at 20 mg/L has highlighted theimportance of producer organisms as a reservoir of antibiotic resistance(D'Costa et al. (2006) Science 311:374). Here bacteria subsisting onantibiotics are described as a substantial addition to the antibioticresistome in terms of both phylogenetic diversity and prevalence ofresistance. The bacteria isolated and described herein are ‘superresistant,’ since they tolerate concentrations of antibiotics>1 g/Lwhich are 50-fold higher than the antibiotic concentrations used todefine the antibiotic resistome. Id.

Greengenes (DeSantis et al. (2006) Applied and EnvironmentalMicrobiology 72:5069) identified isolates among the bacteria subsistingon antibiotics that are closely related to known pathogens e.g., membersof the Burkholderia cepacia complex, and Serratia marcescens. Inprinciple, relatedness allows for easier transfer of genetic material,since codon usage, promoter binding sites and other transcriptional andtranslational motifs are likely to be similar. It is therefore possiblethat pathogenic microbes can more readily use resistance genesoriginating from bacteria subsisting on antibiotics compared to theresistance genes from more distantly related antibiotic producerorganisms.

To date, there have been no reports describing antibiotic catabolism inpathogenic strains. However, since most sites of serious infection inthe human body are not carbon source limited it is unlikely thatpathogenic microbes would have a strong selective advantage bycatabolizing antibiotics compared to just resisting them, so it islikely that only the resistance conferring part of the catabolicmachinery would be selected for in pathogenic strains.

In addition to the finding that bacteria subsisting on natural andsynthetic antibiotics are widely distributed in the environment, theseresults highlight an unrecognized reservoir of multiple antibioticresistance machinery. Bacteria subsisting on antibiotics arephylogenetically diverse, and include many organisms closely related toclinically relevant pathogens. It is thus possible that pathogens couldobtain antibiotic resistance genes from environmentally distributedsuper-resistant microbes subsisting on antibiotics.

REFERENCES

-   Riesenfeld et al. (2004) Environmental Microbiology 6:981-   Walsh (2000) Nature 406:775-   Alekshun and Levy (2007) Cell 128:1037-   Fredrickson et al. (2000) Applied and Environmental Microbiology    66:2006-   McAllister et al. (1996) Biodegradation 7:1-   Kameda et al. (1961) Nature 191:1122-   Abd-El-Malek et al. (1961) Nature 189:775-   Cole et al. (2007) Nucleic Acids Res 35:D169-   Wheeler et al. (2000) Nucleic Acids Res 28:10-   Ludwig et al. (2004) Nucleic Acids Res 32:1363

Example II Materials and Methods

Growth Media

All liquid media used for isolating bacteria capable of subsisting onantibiotics was made by dissolving 1 g/L of the relevant antibiotics(Table 1, which depicts lot purities of antibiotics used, as reported onCertificates of Analysis from Sigma-Aldrich) into single carbon source(SCS) media containing 5 g (NH₄)₂SO₄, 3 g KH₂PO₄, 0.5 g MgSO₄.7H₂O, 15mg EDTA, 4.5 mg ZnSO₄.7H₂O, 4.5 mg CaCl₂.2H₂O, 3 mg FeSO₄.7H₂O, 1 mgMnCl₂.4H₂O, 1 mg H₃BO₃, 0.4 mg Na₂MoO₄.2H₂O, 0.3 mg CuSO₄.5H₂O, 0.3 mgCoCl₂.6H₂O and 0.1 mg KI per liter water. The pH was adjusted to 5.5using HCl, and the media was sterilized through a 0.22 μm filter. Solidmedium was prepared by adding 15 g agar per liter of liquid SCS mediafollowed by autoclaving before adding antibiotics.

TABLE 1 NR, not reported. Antibiotics Lot Purity % Ciprofloxacin 98.5Levofloxacin 100.0 Sisomicin 99 Gentamicin NR Kanamycin NR Amikacin 100Penicillin G 99.7 Carbenicillin 92.9 Dicloxacillin 99.8Chloramphenicol >99 Nalidixic acid 100 Thiamphenicol >99 Sulfisoxazole99.7 Trimethoprim 100 Mafenide 100 Sulfamethizole 99.9 D-Cycloserine 98Vancomycin NR

All liquid media used for resistance profiling was made by dissolving 20mg/L or 1 g/L of the relevant antibiotics into autoclaved Luria brothcontaining 5 g Yeast Extract, 10 g NaCl and 10 g of tryptone in 1 Literof water. The pH was adjusted to 5.5 using HCl, and the media wassterilized through a 0.22 μm filter.

Culturing of Environmental Bacteria Capable of Subsisting on Antibiotics

Initial soil microbial inocula (soil description in Table 2, whichdepicts soil information for the 11 different soils from which bacteriacapable of subsisting on antibiotics were isolated) were prepared inminimal medium containing no carbon, and inoculated into SCS-antibioticmedia (corresponding to approximately 125 mg of dissolved soil in 5 mLof media). To significantly reduce the transfer of residual alternativecarbon sources present in original inocula, samples were passaged (2.5μL) into fresh SCS-antibiotic media (5 mL) two additional times after 7days of growth, resulting in a 5×10⁴ dilution at each passage (resultingin a final carryover of approximately 30 ng of soil in 5 mL of media atthe third passage). Clonal isolates from the liquid cultures wereobtained by plating cultures out on SCS-antibiotic agar medium andresulting single colonies were picked and re-streaked on correspondingplates. Three colonies each were then inoculated into freshSCS-antibiotic liquid media (5 mL) to confirm clonal phenotype. Finalculture growth was recorded after 1 month incubation without shaking at22° C. and cultures with at least 10⁸ cells/mL were assayed as positivegrowth.

TABLE 2 FIG. 1A Soil identifiers Soil type name Soil collection locationF1 Farmland S1G Corn Field with Antibiotic Treated Manure, Great BrookFarm, Carlisle, MA F2 Farmland S1N Alfalfa Field with Manure Treatment,Northcroft Farm, Pelican Rapids, MN F3 Farmland S2N Alfalfa Fieldwithout Manure Treatment, Northcroft Farm, Pelican Rapids, MN P1Pristine S2R Raccoon Ledger, Rockport, MA P2 Pristine S3N Prairie nextto Northcroft Farm, Pelican Rapids, MN P3 Pristine S1R Brier's Swamp,Rockport, MA P4 Pristine S1A Pristine Forest Soil, Alan Seeger NaturalArea, PA P5 Pristine S2T Untreated Forested Area, Toftrees StateGameland Area, PA U1 Urban S1T Waste Water Treated Area, Toftrees StateGameland Area, PA U2 Urban S3F Boston Fens, MA U3 Urban S1P BostonPublic Garden, MA

Since inoculation in media lacking a carbon source (no carbon control)did not show growth in any cases, carbon source contamination of thesource media or carbon fixation from the air were consideredinsignificant to this experiment. The only other alternative carbonsubstrate for growth could be impurities in the antibiotic stocks. Allantibiotics used were purchased from Sigma-Aldrich at the highestpurities available. Lot purities of each compound used are listed inTable 1. Based on an average carbon mass of 0.15×10⁻¹² g per bacterialcell, it was estimated that at least 15 μg of carbon must beincorporated into bacterial biomass to reach sufficient culturedensities in 1 mL of culture to be rated as successful growth. Assuming50% carbon content of impurities, and under the most stringentassumptions of (1) 100% incorporation of carbon impurities into biomass,and (2) no loss of carbon as metabolic byproducts (such as CO₂),antibiotics with greater than 97% purity would have insufficientimpurities to support sole carbon source growth. Of the antibiotic lotsused in this experiment (Table 1), twelve compound stocks are at least99% pure, two compounds (ciprofloxacin and D-cycloserine) have between98 and 98.5% purity, one compound (carbenicillin) is 92.9% pure, and nopurity information is available for three compounds (kanamycin,gentamicin, and vancomycin).

Phylogenetic Profiling

The 16S ribosomal DNA (rDNA) of each of the clonal isolates identifiedin this study was amplified using universal bacterial 16S primers:

>Bact_63f_62C (SEQ ID NO: 1)5′-CAG GCC TAA CAC ATG CAA GTC-3′ >Bact_1389r_63C (SEQ ID NO: 2)5′-ACG GGC GGT GTG TAC AAG-3′

Successful 16S rDNA amplicons were sequenced for phylogenetic profiling.High-quality, non-chimeric sequences were classified using Greengenes(DeSantis et al. (2006) Nucleic Acids Res 34:W394; DeSantis et al.(2006) Applied and Environmental Microbiology 72:5069), with consensusannotations from RDP (Cole et al. (2007) Nucleic Acids Res 35:D169) andNCBI taxonomies (Wheeler et al. (2000) Nucleic Acids Res 28: 10).Phylogenetic trees were constructed using the neighbor joining algorithmin ARB (Ludwig et al. (2004) Nucleic Acids Res 32:1363) using theGreengenes aligned 16S rDNA database. Placement in the tree wasconfirmed by comparing automated Greengenes taxonomy to the annotatedtaxonomies of nearest neighbors of each sequence in the aligneddatabase.

Resistance Profiling of 75 Representative Isolates Capable of Subsistingon Antibiotics

75 clonal isolates (Table 3, which lists strain information for the 75clonal isolates used for resistance profiles) were selected to includemultiple isolates capable of subsisting on each of the 18 antibioticsand originating from each of the 11 soils (Table 2). Bacterial cultureswere inoculated into Luria Broth from frozen glycerol stocks and wereincubated at 22° C. for 3 days. 500 nL of this culture was used toinoculate each of the clonal isolates into 200 uL of Luria Brothcontaining one of the eighteen different antibiotics (See Table 1) at 20mg/L and 1 g/L. Cultures were incubated without shaking at 22° C. for 4days. Resistance of an isolate was determined by turbidity at 600 nmusing a Versamax microplate reader from Molecular Devices.

TABLE 3 FIG. 3A identifier Strain name Subsisting on From soil 1Levo-S2T-M1LLLSSL-2 Levofloxacin S2T 2 Kana-S2T-M1LLLSSL-3 Kanamycin S2T3 Amik-S2T-M1LLLSSL-1 Amikacin S2T 4 Carb-S2T-M1LLLSSL-2 CarbenicillinS2T 5 Chlo-S2T-M1LLLSSL-2 Chloramphenicol S2T 6 Nali-S2T-M1LLLSSL-1Nalidixic acid S2T 7 Thia-S2T-M1LLLSSL-2 Thiamphenicol S2T 8Trim-S2T-M1LLLSSL-1 Trimethoprim S2T 9 Mafe-S2T-M1LLLSSL-3 Mafenide S2T10 Cycl-S2T-M1LLLSSL-3 D-Cycloserine S2T 11 Vanc-S2T-M1LLLSSL-3Vancomycin S2T 12 Siso-S2N-M1LLLSSL-1 Sisomycin S2N 13Gent-S2N-M1LLLSSL-2 Gentamycin S2N 14 Kana-S2N-M1LLLSSL-2 Kanamycin S2N15 Peni-S2N-M1LLLSSL-2 Penicillin G S2N 16 Dicl-S2N-M1LLLSSL-1Dicloxacillin S2N 17 Trim-S2N-M1LLLSSL-1 Trimethoprim S2N 18Vanc-S2N-M1LLLSSL-1 Vancomycin S2N 19 Dicl-S3N-M1LLLSSL-2 DicloxacillinS3N 20 Thia-S3N-M1LLLSSL-3 Thiamphenicol S3N 21 Trim-S3N-M1LLLSSL-2Trimethoprim S3N 22 Mafe-S3N-M1LLLSSL-2 Mafenide S3N 23Vanc-S3N-M1LLLSSL-2 Vancomycin S3N 24 Cipr-S1P-M1LLLSSL-3 CiprofloxacinS1P 25 Peni-S1P-M1LLLSSL-2 Penicillin G S1P 26 Chlo-S1P-M1LLLSSL-1Chloramphenicol S1P 27 Thia-S1P-M1LLLSSL-1 Thiamphenicol S1P 28Trim-S1P-M1LLLSSL-3 Trimethoprim S1P 29 Slfm-S1P-M1LLLSSL-2Sulfamethizole S1P 30 Cycl-S1P-M1LLLSSL-1 D-Cycloserine S1P 31Vanc-S1P-M1LLLSSL-3 Vancomycin S1P 32 Cipr-S1T-M1LLLSSL-2 CiprofloxacinS1T 33 Levo-S1T-M1LLLSSL-1 Levofloxacin S1T 34 Siso-S1T-M1LLLSSL-1Sisomycin S1T 35 Carb-S1T-M1LLLSSL-1 Carbenicillin S1T 36Dicl-S1T-M1LLLSSL-1 Dicloxacillin S1T 37 Chlo-S1T-M1LLLSSL-1Chloramphenicol S1T 38 Thia-S1T-M1LLLSSL-3 Thiamphenicol S1T 39Trim-S1T-M1LLLSSL-2 Trimethoprim S1T 40 Mafe-S1T-M1LLLSSL-1 Mafenide S1T41 Cycl-S1T-M1LLLSSL-2 D-Cycloserine S1T 42 Vanc-S1T-M1LLLSSL-1Vancomycin S1T 43 Levo-S3F-M1LLLSSL-3 Levofloxacin S3F 44Slfs-S3F-M1LLLSSL-3 Sulfisoxazole S3F 45 Trim-S3F-M1LLLSSL-lTrimethoprim S3F 46 Mafe-S3F-M1LLLSSL-3 Mafenide S3F 47Slfm-S3F-M1LLLSSL-3 Sulfamethizole S3F 48 Vanc-S3F-M1LLLSSL-2 VancomycinS3F 49 Amik-S1R-M1LLLSSL-3 Amikacin S1R 50 Peni-S1R-M1LLLSSL-2Penicillin G S1R 51 Mafe-S1R-M1LLLSSL-2 Mafenide S1R 52Vanc-S1R-M1LLLSSL-2 Vancomycin S1R 53 Trim-S1N-M1LLLSSL-1 TrimethoprimS1N 54 Vanc-S1N-M1LLLSSL-1 Vancomycin S1N 55 Kana-S1A-M1LLLSSL-2Kanamycin S1A 56 Carb-S1A-M1LLLSSL-2 Carbenicillin S1A 57Slfs-S1A-M1LLLSSL-1 Sulfisoxazole S1A 58 Vanc-S1A-M1LLLSSL-2 VancomycinS1A 59 Kana-S2R-M1LLLSSL-2 Kanamycin S2R 60 Amik-S2R-M1LLLSSL-3 AmikacinS2R 61 Peni-S2R-M1LLLSSL-2 Penicillin G S2R 62 Dicl-S2R-M1LLLSSL-1Dicloxacillin S2R 63 Mafe-S2R-M1LLLSSL-2 Mafenide S2R 64Slfm-S2R-M1LLLSSL-1 Sulfamethizole S2R 65 Cipr-S1G-M1LLLSSL-1Ciprofloxacin S1G 66 Levo-S1G-M1LLLSSL-1 Levofloxacin S1G 67Gent-S1G-M1LLLSSL-3 Gentamycin S1G 68 Kana-S1G-M1LLLSSL-1 Kanamycin S1G69 Peni-S1G-M1LLLSSL-1 Penicillin G S1G 70 Carb-S1G-M1LLLSSL-3Carbenicillin S1G 71 Chlo-S1G-M1LLLSSL-3 Chloramphenicol S1G 72Nali-S1G-M1LLLSSL-2 Nalidixic acid S1G 73 Thia-S1G-M1LLLSSL-1Thiamphenicol S1G 74 Slfs-S1G-M1LLLSSL-3 Sulfisoxazole S1G 75Mafe-S1G-M1LLLSSL-2 Mafenide S1G

Analysis of Antibiotic Removal of Penicillin and CarbenicillinSubsisting Bacteria

Representative isolates capable of growth on penicillin andcarbenicillin as sole carbon source were selected for analysis ofantibiotic removal from the growth media by High Performance LiquidChromatography (HPLC). 2 μL of these cultures were re-inoculated intofresh SCS-antibiotic medium (5 mL) and allowed to grow for 28 days.Samples of the cultures and un-inoculated media controls were taken atregular intervals throughout the 28 day period and the catabolism ofpenicillin and carbenicillin was monitored at 214 nm by HPLC of filteredmedia from samples using a Hewlett Packard 1090 Liquid Chromatograph anda Vydac C-18 column. HPLC was performed at a flow rate of 0.3 mL/minwith an acetonitrile gradient going from 5% to 65% in 30 minutes in thepresence of 0.1% trifluoroacetic acid.

The HPLC chromatogram of the penicillin catabolizing culture medium(FIG. 1B) started out with a single peak corresponding to the penicillinpeak of the un-inoculated control. This peak disappeared at day 4 withthe appearance of multiple smaller peaks at lower elution times; by day20 these peaks had also disappeared in agreement with the completecatabolism of penicillin by the culture in 20 days. In comparison, thesingle penicillin peak in the un-inoculated control remained thedominant peak over the same time course. The HPLC chromatogram of themedium from the carbenicillin catabolizing culture (FIG. 1B) started outwith a bimodal peak corresponding to the un-inoculated carbenicillincontrol, which remained stable for 2 days. At day 4, corresponding tothe appearance of visible turbidity in the inoculated culture, thebimodal peak had almost disappeared and secondary peaks at lower elutiontimes were observed. These secondary peaks almost completely disappearedby the 28^(th) day, suggesting that carbenicillin was almost completelycatabolized within 28 days. The bimodal carbenicillin peak remainedrelatively unchanged in the un-inoculated control over the same timecourse.

Samples from the penicillin subsisting culture from day 0 and day 4 wereprepared for LC/MS using a Waters Sep-Pak Cartridge prior to massspectrometry analysis using a LTQ-FT from Thermo Electron. Mass spectrawere analyzed using XCalibur 2.0.5 and the empirically determined m/zvalues of all major peaks were compared to predicted m/z values ofputative penicillin degradation products calculated using ChemDraw Ultra9.0 (FIGS. 6A-6C).

What is claimed is:
 1. A clonal isolate of a bacterium that can use oneor more antibiotics as a carbon source, wherein the bacterium has a 16Snucleic acid sequence comprising a GenBank Accession Number selectedfrom the group consisting of EU515334, EU515335, EU515336, EU515337,EU515338, EU515339, EU515400, EU515401, EU515402, EU515403, EU515404,EU515405, EU515406, EU515407, EU515408, EU515409, EU515410, EU515411,EU515412, EU515413, EU515414, EU515415, EU515416, EU515417, EU515418,EU515419, EU515420, EU515421, EU515422, EU515423, EU515424, EU515425,EU515426, EU515427, EU515428, EU515429, EU515430, EU515431, EU515432,EU515433, EU515434, EU515435, EU515436, EU515437, EU515438, EU515439,EU515440, EU515441, EU515442, EU515443, EU515444, EU515445, EU515446,EU515447, EU515448, EU515449, EU515450, EU515451, EU515452, EU515453,EU515454, EU515455, EU515456, EU515457, EU515458, EU515459, EU515460,EU515461, EU515462, EU515463, EU515464, EU515465, EU515466, EU515467,EU515468, EU515469 EU515470, EU515471, EU515472, EU515473, EU515474,EU515475, EU515476, EU515477, EU515478, EU515479, EU515480, EU515481,EU515482, EU515483, EU515484, EU515485, EU515486, EU515487, EU515488,EU515489, EU515490, EU515491, EU515492, EU515493, EU515494, EU515495,EU515496, EU515497, EU515498, EU515499, EU515500, EU515501, EU515502,EU515503, EU515504, EU515505, EU515506, EU515507, EU515508, EU515509,EU515510, EU515511, EU515512, EU515513, EU515514, EU515515, EU515516,EU515517, EU515518, EU515519, EU515520, EU515521, EU515522, EU515523,EU515524, EU515525, EU515526, EU515527, EU515528, EU515529, EU515530,EU515531, EU515532, EU515533, EU515534, EU515535, EU515536, EU515537,EU515538, EU515539, EU515540, EU515541, EU515542, EU515543, EU515544,EU515545, EU515546, EU515547, EU515548, EU515549, EU515550, EU515551,EU515552, EU515553, EU515554, EU515555, EU515556, EU515557, EU515558,EU515559, EU515560, EU515561, EU515562, EU515563, EU515564, EU515565,EU515566, EU515567, EU515568, EU515569, EU515570, EU515571, EU515572,EU515573, EU515574, EU515575, EU515576, EU515577, EU515578, EU515579,EU515580, EU515581, EU515582, EU515583, EU515584, EU515585, EU515586,EU515587, EU515588, EU515589, EU515590, EU515591, EU515592, EU515593,EU515594, EU515595, EU515596, EU515597, EU515598, EU515599, EU515600,EU515601, EU515602, EU515603, EU515604, EU515605, EU515606, EU515607,EU515608, EU515609, EU515610, EU515611, EU515612, EU515613, EU515614,EU515615, EU515616, EU515617, EU515618, EU515619, EU515620, EU515621,EU515622 and EU515623.
 2. The bacterium of claim 1, wherein one or moreantibiotics are from the antibiotic class selected from the groupconsisting of pyrimidine derivative, sulfonamide, quinolone,glycopeptides, beta-lactam, amphenicols, aminoglycoside and amino acidderivative.
 3. The bacterium of claim 1, wherein one or more antibioticsare selected from the group consisting of chloramphenicol, penicillin G,vancomycin, carbenicillin, ciprofloxacin, mafenide, kanamycin,sisomicin, amikacin, trimethropin, D-cycloserine, gentamicin,dicloxacillin, nalidixic acid, thiamphenicol, levofloxacin,sulfamethizole and sulfisoxazole.
 4. The bacterium of claim 1, whereinthe bacterium uses the one or more antibiotics as a sole carbon source.